CLONING VECTORS FOR E.COLI BASED ON E.COLI PLASMIDS Different types Principle Nguyen Thu Giang-IPMB, VUB
CLONING VECTORS FOR E.COLI
BASED ON E.COLI PLASMIDS
Different types
Principle
Nguyen Thu Giang-IPMB, VUB
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What is a plasmid?
• Plasmids are small, extrachromosomal pieces of bacterial DNA that are often antibiotic resistant
• They are „shuttle vectors‟ to create, produce,
and maintain recombinant DNA
• An example of the first cloning vector for
E.coli based on E.coli plasmids is pBR322
And pUC18 now is the most commonly used
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Characters
• A way to maintain in engineered DNA in
the cell’s progeny
• A way to identify (or select) the cells
containing DNA
• A place to insert the gene or piece of
DNA you want to work with
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General properties of plasmid
vectors:
• small, easily isolated, ease of transformation
• high copy number
• polylinkers or multiple cloning sites (MCS)
• selection for transformation (drug resistance)
• ease of screening (lacZ)
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Specific properties of plasmid
vectors:
• they must to appropriate to the host, e.g. ori, selection
• they must be appropriate to your objectives
- expression or non-expression
- expression of RNA or protein
- integration and non-integration
- low or high copy number
- ease of sequencing
- facilitate library construction (“the size problem”)
- work in different species (shuttle vectors)
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pBR322
• Nomenclature
– ‘p’ indicates that it is indeed a plasmid
– ‘BR’ identified the laboratory in which the
vector was originally constructed
– ‘322’ distinguishes this plasmid from others
developed in the same laboratory
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pBR322
• pBR322 is an example of an early
cloning vector that replicates in E.coli.
• pBR322 has some significant features:
– It has a small size of 4363 bp
– It carries two sets of antibiotic resistance
genes
– It has reasonably high copy number
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pBR322• Ori allows independent
replication
• Single cleavage sites for various restriction enzyme (BamHI ect)
• tet resistance gene
• amp resistance gene
• Mechanism:• insertion of foreign DNA
at BamHI site
• tet resistance gene inactivated
• transformants carrying foreign DNA are amp resistant but tetracycline sensitive
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pBR322
• The pedigree
– amp gene
originally resided
on the plasmid
R1
– tet gene is
derived from R6-5
– The replication
origin of pBR322
is originally from
pMB1
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“Evolution” of Plasmid Vectors
pUC series
pGEM series
Protein Purification
pGEX series
Regulated Expression
pET, pBAD, pTET
CosmidsPhagmid
λZAP
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pBR327
• pBR327, one of the first derived from
pBR322, was produced by removing
1089 bp from pBR322:
– pBR327 has a higher copy number
– pBR327 is non-conjugative
Both are now no longer widely used
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pUC series
• next generation, although only replication origin and amp genes remain
• used in BIO lab• collects all restrictions sites into MCS or polylinker
• 3 advantages:– fortuitous: the manipulations involved in construction
of pUC series were accompanied by a chance mutation
– Identification of recombinant cells can be achieved by a single step process and pUC series introduces screening with x-gal
– The clustering of restriction sites allows DNA fragments with two sticky ends.
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pGEM3Z
• next generation,
2750bp
• very similar to pUC
vectors but has two
promoters
• introduces RNA
expression for in vitro
or in vivo expression.
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CLONING PROCESS
• Gene of interest is cut out with RE
• Host plasmid is cut with same RE
• Gene is inserted into plasmid and ligated with ligase
• New plasmid inserted into bacterium (transform)
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PLASMID CLONING
STRATEGY
• Involves five steps:
1.Enzyme restriction digest of DNA sample.
2.Enzyme restriction digest of DNA plasmid vector.
3.Ligation of DNA sample products and plasmid vector.
4.Transformation with the ligation products.
5.Growth on agar plates with selection for antibiotic resistance.
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STEP 4. TRANSFORMATION OF
LIGATION PRODUCTS
• The process of transferring exogenous DNA into
cells is call “transformation”
• There are basically two general methods for
transforming bacteria. The first is a chemical
method utilizing CaCl2 and heat shock to
promote DNA entry into cells.
• A second method is called electroporation based
on a short pulse of electric charge to facilitate
DNA uptake.
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STEP 5
• Blue colonies represent Ampicillin-resistant bacteria
that contain pVector and express a functional alpha
fragment from an intact LacZ alpha coding
sequence.
White colonies represent Ampicillin-resistant
bacteria that contain pInsert and do not produce
LacZ alpha fragment